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Hexameric Ring Structure of the N-Terminal Domain of Mycobacterium Tuberculosis Dnab Helicase Tapan Biswas and Oleg V

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Hexameric Ring Structure of the N-Terminal Domain of Mycobacterium Tuberculosis Dnab Helicase Tapan Biswas and Oleg V Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase Tapan Biswas and Oleg V. Tsodikov Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA Keywords Hexameric DnaB helicase unwinds the DNA double helix during replica- crystal structure; DnaB helicase; primase; tion of genetic material in bacteria. DnaB is an essential bacterial protein; replication; tuberculosis therefore, it is an important potential target for antibacterial drug disco- very. We report a crystal structure of the N-terminal region of DnaB from Correspondence ˚ O. V. Tsodikov, Department of Medicinal the pathogen Mycobacterium tuberculosis (MtDnaBn), determined at 2.0 A Chemistry, College of Pharmacy, University resolution. This structure provides atomic resolution details of formation of Michigan, Ann Arbor, MI 48109-1065, of the hexameric ring of DnaB by two distinct interfaces. An extensive USA hydrophobic interface stabilizes a dimer of MtDnaBn by forming a four- Fax: +1 734 647 8430 helix bundle. The other, less extensive, interface is formed between the Tel: +1 734 936 2676 dimers, connecting three of them into a hexameric ring. On the basis of E-mail: [email protected] crystal packing interactions between MtDnaBn rings, we suggest a model (Received 19 March 2008, revised 8 April of a helicase–primase complex that explains previously observed effects of 2008, accepted 11 April 2008) DnaB mutations on DNA priming. doi:10.1111/j.1742-4658.2008.06460.x The DnaB gene encoding DnaB helicase was originally required for formation of the functional helicase identified in a region of the bacterial genome, mutations [11,12] and for binding DnaG [13], and a C-terminal in which caused a defect in DNA replication at elevated RecA-like domain, responsible for DNA binding and temperatures in vivo [1]. DnaB is an ATP-dependent ATP hydrolysis (Fig. 1A). Six monomers of DnaB helicase that unwinds parental duplex DNA during form a two-tiered ring, as observed in recent modest- replication [2–5], thereby allowing the two strands to be resolution structures of DnaB from the thermophilic copied. A hexamer of DnaB encircles the lagging strand Geobacillus stearothermophilus (Gs) [14], and in a low- and moves on it in the 5¢fi3¢ direction, thereby split- resolution structure of a DnaB homolog G40P from ting the two complementary DNA strands [6]. A hexa- Bacillus subtilis phage SPP1 [12], reported while this meric DnaB helicase alone is not very processive [7], article was in preparation. One tier is formed by whereas it becomes highly processive when bound to DnaBn, which has three-fold symmetry. The other tier other replication proteins [8]. During DNA replication, is formed by the RecA-like ATPase domains in DnaB is associated with multiple copies of DNA prim- approximate six-fold symmetry. The hexameric compo- ase DnaG, which catalyzes the formation of RNA prim- sition of DnaB is shared by other representatives of ers for the synthesis of Okazaki fragments on the the same superfamily of replicative helicases, such as lagging strand. The DnaB–DnaG complex plays a key bacteriophage T7 gene 4 protein [15], although sig- role in coordinating the mechanistically different contin- nificant differences in the sequence and domain orga- uous and discontinuous DNA syntheses on the leading nization exist among the family members. and the lagging strands, respectively [9]. DnaB helicase is conserved in the eubacterial king- A monomer of DnaB consists of two domains [10]: dom, and is distinct from its functional relatives an N-terminal helical domain (DnaBn), shown to be in higher eukaryotes. Therefore, DnaB is a very Abbreviations DnaBn, N-terminal helical domain of DnaB helicase; DnaGc, C-terminal domain of DnaG; Gs, Geobacillus stearothermophilus; HhH, helix– hairpin–helix (HhH); Mt, Mycobacterium tuberculosis. 3064 FEBS Journal 275 (2008) 3064–3071 ª 2008 The Authors Journal compilation ª 2008 FEBS T. Biswas and O. V. Tsodikov Mycobacterium tuberculosis DnaB helicase Fig. 1. (A) Domain organization of MtDnaB. The numbers indicate approximate domain boundaries. The C-terminal domain of DnaB in M. tuberculosis and several other mycobacteria is disrupted by an intein, as indicated. (B) Sequence alignment of DnaBn. Sequences of DnaBn from several bacterial species (Mt, M. tuberculosis; Ml, Mycobacterium leprae; Ms, Mycobacterium smegmatis; Gs, G. stearother- mophilus; Ec, E. coli) are aligned with the secondary structures of MtDnaBn (above; this study) and GsDnaBn [14]. The residues in the HhH–HhH interface of MtDnaBn hexamer are indicated by black bars, and those in the dimer–dimer interface are indicated by black circles. The surface residues of MtDnaB that form the putative DnaG-binding pocket (this study) are indicated by open red circles, and those of GsDnaB that contact DnaG in the GsDnaB–GsDnaG structure [14] are indicated by filled red circles. (C) SDS ⁄ PAGE gel of purified MtDnaB(21–197) and MtDnaB(21–134) proteins. (D) A gel filtration chromatogram demonstrating that MtDnaB(21–197) elutes as a dimer and MtDnaB(21–134) as a monomer. attractive target for antibacterial therapy. The C-ter- We report a 2.0 A˚resolution structure of the hexa- minal catalytic domain of DnaB is very highly con- meric ring DnaBn from the pathogen Mycobacte- served, which points at the conservation of the rium tuberculosis (MtDnaBn). On the basis of the ATPase function in this helicase superfamily. DnaBn organization of the MtDnaBn rings in the crystal is less conserved, and its binding partner, the C-ter- lattice, we propose a model of the mycobacterial minal domain of primase DnaG (DnaGc), is very DnaB–DnaG complex. weakly conserved. This divergence is intriguing, because it must be related to documented differences Results and Discussion in intrinsic stabilities of DnaB–DnaG complexes between Escherichia coli and G. stearothermophilus Structure of the hexameric ring of MtDnaBn [13,16–18]. This, in turn, implies divergent coordina- tion of unwinding with priming at the replication Bacterial DnaB helicase consists of two domains: the fork in different bacteria. moderately conserved, entirely helical DnaBn ( 24% FEBS Journal 275 (2008) 3064–3071 ª 2008 The Authors Journal compilation ª 2008 FEBS 3065 Mycobacterium tuberculosis DnaB helicase T. Biswas and O. V. Tsodikov identity) (Fig. 1B) and the highly conserved C-terminal one of the projections of electron microscopic structures ATPase domain ( 40% identity [19]). The two of full-length E. coli DnaB hexamer [21]. Three mono- domains are connected by a linker region (Fig. 1A). In mers, one from each dimer, form the inner rim, and the M. tuberculosis DnaB, MtDnaBn (residues 21–197, other three monomers form the outer rim of the triangle. including the linker) forms a stable dimer in solution The backbones of all six monomers are generally very (Fig. 1C,D). The region spanning residues 135–197 is similar. There are subtle differences in the conforma- required for dimer stability, because a truncated form of tions of C-terminal helices a7 and a8 and in the hairpins the protein (residues 21–134) is monomeric (Fig. 1C,D). connecting them (not shown). These differences indicate MtDnaBn forms a hexamer in the crystallization solu- a small degree of conformational plasticity of the region tion; the crystals of DnaBn contain one slightly asym- closest to the mobile C-terminal domain of DnaB [14]. metric hexamer per asymmetric unit. Three two-fold The interface stabilizing each dimer is formed symmetrical dimers of DnaBn are assembled into a hex- between the C-terminal helix–hairpin–helix (HhH) amer that has approximate three-fold rotational symme- regions of DnaBn (residues 128–165) that protrude try (Fig. 2A). The two-fold and the three-fold symmetry from the globular subdomains (residues 21–127) axes are parallel to each other. The hexamer forms an (Fig. 2B). This explains our observation of the mono- opening in the shape of an equilateral triangle with a meric state of truncated DnaBn (residues 21–134). This side length of 51 A˚. Therefore, the ring can accommo- interface is composed of conserved interdigitating date a cylinder with a maximum diameter of 34 A˚, large hydrophobic residues (Figs 1B and 2B) that bury enough for duplex and even triplex DNA. Indeed, DnaB 2200 A˚2 of solvent-accessible surface area [22]. The is able to encircle and actively translocate on duplex Ca atoms of two glycine residues, Gly136 and Gly143, DNA [20]. The triangular shape is roughly similar to contribute to this interface and allow the helices Fig. 2. The structure of the MtDnaBn hex- amer. (A) The hexameric ring of MtDnaBn is shown as a ribbon diagram. (B) The HhH–HhH interface. (C) The dimer–dimer interface. In (B) and (C), the residues that form the respective interfaces (see also Fig. 1B) are shown as sticks, with a subset of them labeled. 3066 FEBS Journal 275 (2008) 3064–3071 ª 2008 The Authors Journal compilation ª 2008 FEBS T. Biswas and O. V. Tsodikov Mycobacterium tuberculosis DnaB helicase to pack snugly against each other. DnaBs of other an inner monomer and the HhH subdomain of an outer bacteria contain either a Gly or an Ala at these monomer. Mutations in this interface significantly two positions. Previously reported mutations in reduce the helicase activity of the DnaB homolog G40P G. stearothermophilus [23] and Salmonella typhimurium [12]. The residues in the dimer–dimer interface are [24] DnaB that cause a priming defect (Val133 and generally, but not universally, conserved (Fig. 1B). Val139 in MtDnaB) lie in this hydrophobic interface. The inner surface of the DnaBn ring contains three These substitutions are likely to abolish DnaB–DnaG positively charged patches, each formed by Arg90, binding by disrupting the structure of DnaBn. The sol- Arg91, Arg 95, and Arg96 (Fig. 3A), that may bind vent-exposed residues of the HhH are generally not the lagging DNA strand during replication.
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